RNA Detection

amplification curve calculated in the previous step. Adjust the

amount of Input and IP cDNA template so that the same

number of PCR cycles can be applied (seeNote 10).

6. Purify the cDNA from the PCR mixture using Agencourt
   AMPure XP Beads following the manufacturer’s instructions.


7. Analyze RNA size on an Agilent 2100 Bioanalyzer. Ensure that
   fragments are 200–250 bp in length, and that few or no dimers
   are present (dimers are seen at ~127 bp).


8. If the Bioanalyzer analysis indicates that primer dimers are
   present, perform size selection using gel electrophoresis:


9. Run the PCR product on a 2.5% low melting point agarose gel
   with 0.5μg/mL ethidium bromide in 1TAE buffer at 90 V
   for 40 min along with the DNA marker provided in the Illu-
   mina kit.


10. Use a transilluminator to image the gel. Use a clean, sharp
    scalpel to isolate the higher band around 200–250 bp, which
    contains the cDNA library, being careful to avoid the lower
    band at 127 bp, which consists of primer dimers.


11. Weigh the gel fragment in a colorless tube, and add 3 volumes
    of Buffer QG from the MinElute Gel Extraction Kit to the gel
    slice.


12. Let the gel slice completely dissolve in the Buffer QG at room
    temperature (~10 min).


13. Isolate the cDNA library by following the remaining steps of
    the MinElute Gel Extraction Kit manufacturer’s protocol.


14. Elute with 10μL of DEPC-treated nuclease-free water (see
    Note 11).


15. Deep sequence the cDNA library using an Illumina HiSeq
    platform (or similar).

4 Notes

1. Although the RNeasy Plus Mini Kit is an efficient method to
   purify total RNA from limited samples of animal cells or tissues,
   it isolates only RNA molecules longer than 200 nt. This proce-
   dure enriches mRNA species, since RNAs below the< 200
   nucleotide cutoff, such as miRNAs, tRNAs, and smaller
   rRNAs, are excluded. If the user wishes to analyze RNA species
   shorter than 200 nt, an alternative is to use the Direct-zol™
   RNA Miniprep Plus kit (Zymo Research)with on-column
   DNAse I digestion, following the manufacturer’s protocol.


2. Do not detach the cells from the plate using trypsin, as this may
   compromise RNA integrity. If using easily detached cells, such

Identifying the m^6 A Methylome 55